Currently, available technologies for the attachment of 5′ end labels to synthetic oligonucleotides rely on two general approaches. The most popular approach requires the production of phosphoramidite derivatives of the desired label. In general, these phosphoramidites are of the structure L-LA-CEP where L is the desired label, LA is a linker arm and CEP is the protected phosphoramidite portion of the oligonucleotide molecule.
The resulting phosphoramidites are then coupled to the synthetic oligonucleotide via standard automated procedures. This process suffers from a number of disadvantages. First, the desired phosphoramidites are typically produced via multi-step, linear syntheses, making this a costly process. Second, phosphoramidites are compounds of limited stability resulting in significant losses when the compounds are purified and limited shelf life, particularly in solution.
The second approach is used in cases where no phosphoramidite is available or where the desired label is not compatible with standard DNA synthesis or deprotection methodologies. In this instance, a suitably protected linker arm phosphoramidite is attached via standard DNA synthesis procedures. Following cleavage and deprotection of the modified oligonucleotide, the label is added to the linker arm in a solution phase reaction. Typically this is accomplished via coupling of an activated ester form of the label to a terminal amine on the linker arm. As before, there are significant disadvantages to this approach. Solution phase chemistry is more labor intensive than traditional solid phase approaches making it more costly and resulting in lower yields. As with phosphoramidites, suitable activated labels must be synthesized or licensed. However, this approach has an advantage in that a common set of linker-CEPs can be utilized with a number of potential labels.
While existing technologies provide a way to synthesize oligonucleotides containing a number of standard labels for research purposes, what is lacking is a generalized procedure for the rapid production of oligonucleotides with novel labels and the economical synthesis of oligonucleotides with standard labels.